Ricin RGA60:: Evidence of its phospholipase activity

The present work documents, on a qualitative and quantitative basis, the lipolytic activity of ricin protein RCA(60) on glycerophospholipids. RCA(60) demonstrates a low level of hydrolysis towards radioactive dipalmitoyl-glycerophosphatidylcholine. This observation was confirmed on a better substrate, palmitoyloleoyl-glycerophosphatidylcholine, after analysis of the reaction products by thin-layer and gas chromatography, A comparable hydrolytic activity was observed when palmitoyl-oleoyl-glycerophosphatidylethanolamine was used as substrate. The nature of the hydrolysis products supports the conclusion that RCA(60) demonstrates phospholipase A(1) and A(2) activities as well as a lysophospholipase activity of A(1) and A(2) type. The insensitivity of this lipolytic activity towards calcium ions and the presence of the already described consensus sequence of lipases, Gly-Xaa-Ser-Xaa-Gly, in the primary sequence of the B-chain of RCA,, support the idea that the lipolytic activity of RCA,, is more related to the lipase family than to the phospholipases A. We hypothesize that such activity contributes to the mechanism which underlies the expression of the cytotoxicity of RCA(60). (C) 1999 Academic Press.